Recombinant super-compound interferon

ABSTRACT

This invention provides a recombinant super-compound interferon or an equivalent thereof with changed spatial configuration. The super-compound interferon possesses anti-viral or anti-tumor activity and therefore is useful to prevent and treat viral diseases and cancers. This invention also provides an artificial gene which codes for the super-compound interferon or its equivalent. Finally, this invention provides methods to produce recombinant super-compound interferon or its equivalent and various uses of said interferon.

The application is a continuation-in-part application of InternationalPatent Application No. PCT/CN02/00128, filed on 28 Feb. 2002, whichclaims priority of Chinese Application No. 01104367.9, filed on 28 Feb.2001, the contents of which are incorporated by reference here into thisapplication.

Throughout this application, various references are referred to.Disclosures of these publications in their entireties are herebyincorporated by reference into this application in order to more fullydescribe the state of the art to which this invention pertains.

FIELD OF THE INVENTION

This invention is related to a recombinant super-compound interferon(rSIFN-co) with changed spatial configuration. One characteristic ofrSIFN-co in this invention is that it cannot only inhibit DNA(deoxyribonucleic acid) duplication of the hepatitis B virus but alsothe secretion of HBsAg and HBeAg.

BACKGROUND OF THE INVENTION

rSIFN-co is a new interferon molecule constructed with the most popularconservative amino acid found in natural human α-IFN subtypes usinggenetic engineering methods. U.S. Pat. Nos. 4,695,623 and 4,897,471 havedescribed it. rSIFN-co had been proved to have broad-spectrum IFNactivity and virus- and tumor-inhibition and natural killer cellactivity. U.S. Pat. No. 5,372,808 by Amgen, Inc. addresses treatmentrSIFN-co. Chinese Patent No. 97193506.8 by Amgen, Inc. addressesre-treatment of rSIFN-co on hepatitis C. Chinese Patent No. 98114663.5by Shenzhen Jiusheng Bio-engineering Ltd. addresses treatment ofrSIFN-co on hepatitis B and hepatitis C.

The United States Food and Drug Administration (FDA) authorized Amgen toproduce rSIFN-co with E. Coli. for clinical hepatitis C treatment at theend of 1997.

Hepatitis B patients can be identified when detecting HBsAg and theHBeAg. α-IFN is commonly used in clinics to treat hepatitis B. IFN bindssuperficial cell membrane receptors, inhibiting DNA and RNA (ribonucleicacid) duplication, including inducing some enzymes to preventduplication of the virus in hepatitis-infected cells. All IFNs caninhibit only the DNA duplication of viruses, not the e and s antigen.

This disclosure describes recombinant super-compound interferon, methodto produce the same and uses thereof.

SUMMARY OF THE INVENTION

This invention provides a recombinant super-compound interferon or anequivalent thereof with changed spatial configuration. An equivalent isa molecule which is similar in function to the super-compoundinterferon. The super-compound interferon possesses anti-viral oranti-tumor activity. This invention also provides an artificial genecodes for the super-compound interferon or its equivalent.

This invention provides a process for production of recombinantsuper-compound interferon comprising introducing an artificial gene withselected codon preference into an appropriate host, culturing saidintroduced host in an appropriate condition permitting expression ofsaid super-compound interferon and harvesting the expressedsuper-compound interferon.

This invention provides a composition comprising the recombinantsuper-compound interferon or its equivalent and a suitable carrier. Thisinvention further provides a pharmaceutical composition comprising therecombinant super-compound interferon or its equivalent and apharmaceutically acceptable carrier.

This invention provides a method for treating viral diseases or tumor ina subject comprising administering to the subject an effective amount ofthe super-compound interferon or its equivalent.

This invention provides the above-described method whereinsuper-compound interferon was administered via oral, vein injection,muscle injection, peritoneal injection, subcutaneous injection, nasal,mucosal administration, by inhalation via an inspirator.

DETAILED DESCRIPTION OF THE FIGURES

FIG. 1. rSIFN-co cDNA sequence designed according to E. Coli. codonusage and deduced rSIFN-co amino acid sequence

FIG. 2. Sequence of another super-compound interferon FIG. 3. Diagram ofpLac T7 cloning vector plasmid

FIG. 4. Diagram of pHY-4 expression vector plasmid

FIG. 5. Construction process of expression plasmid pHY-5

FIG. 6-A. Circular Dichroism spectrum of Infergen®

Spectrum range: 250 nm-190 nmSensitivity: 2 m°/cmLight path: 0.20 cm

Equipment: Circular Dichroism J-500C

Samples: contains 30 μg/ml IFN-con1, 5.9 mg/ml of NaCl and 3.8 mg/ml ofNa₂PO₄, pH7.0.Infergen® (interferon alfacon-1), made by Amgen Inc., also known asconsensus interferon, is marketed for the treatment of adults withchronic hepatitis C virus (HCV) infections. It is currently the only FDAapproved, bio-optimized interferon developed through rational drugdesign and the only interferon with data in the label specifically fornon-responding or refractory patients. InterMune's sales forcere-launched Infergen® in January 2002 with an active campaign to educateU.S. hepatologists about the safe and appropriate use of Infergen®,which represents new hope for the more than 50 percent of HCV patientswho fail other currently available therapies. Seehttp://www.intermune.com/wt/itmn/infergen, Aug. 27, 2003

FIG. 6-B. Circular Dichroism spectrum of Infergen® From Reference[Journal of Interferon and Cytokine Research. 16:489-499 (1996)]

FIG. 6-C. Circular Dichroism spectrum of rSIFN-co

Spectrum range: 320 nm-250 nmSensitivity: 2 m°/cmLight path: 2 cm

Equipment: Circular Dichroism J-500C

Samples: contains 0.5 mg/ml rSIFN-co, 5.9 mg/ml of NaCl and 3.8 mg/ml ofNa₂PO₄, pH7.0.

FIG. 6-D. Circular Dichroism spectrum of rSIFN-co

Spectrum range: 250 nm-190 nmSensitivity: 2 m°/cmLight path: 0.20 cm

Equipment: Circular Dichroism J-500C

Samples: contains 30 μg/ml rSIFN-co, 5.9 mg/ml of NaCl and 3.8 mg/ml ofNa₂PO₄, pH7.0.Clearly, as evidenced by the above spectra, the secondary or eventertiary structure of rSIFN-co is different from Infergen®.

DETAILED DESCRIPTION OF THE INVENTION

This invention provides a recombinant super-compound interferon or anequivalent thereof with changed spatial configuration. This inventionreveals that protein with same primary sequence might have differentbiological activities. As illustrated in the following example, thisinvention disclosed two proteins with identical amino acid sequence butwith different activities. This activity may sometimes become improvedefficacy and sometimes, the protein with changed spatial configurationwould reveal new function.

An equivalent is a molecule which is similar in function to the compoundinterferon. An equivalent could be a deletion, substitution, orreplacement mutant of the original sequence. Alternatively, it is alsothe intention of this invention to cover mimics of the recombinantsuper-compound interferon. Mimics could be a peptide, polypeptide or asmall chemical entity.

The interferon described herein includes but is not limited tointerferon α, β, or ω. In an embodiment, it is IFN-1a, IFN-2b or othermutants.

In an embodiment, the super-compound interferon disclosed has higherefficacy than the interferon described in U.S. Pat. No. 4,695,623 or4,897,471. This super-compound interferon is believed to have uniquesecondary or tertiary structure. (See e.g. FIG. 6)

The super-compound interferon described herein has spatial structurechange(s) resulting from the changes of its production process.

The above-described super-compound interferon may be produced by a highefficiency expression system which uses a special promoter. In anembodiment, the promoter is P_(BAD). As it could be easily appreciatedby other ordinary skilled artisan, other inducible promoter, such asheat shock promoter, may be used in this invention.

The super-compound interferon may also be produced with its gene asartificially synthesized cDNA with adjustment of its sequence from thewild-type according to codon preference of E. Coli. Extensive discussionof said codon usage (preference) may be found in U.S. Pat. No.4,695,623. See e.g. column 6, line 41—column 7, line 35

The above described super-compound interferon possesses anti-viral oranti-tumor activity and therefore useful in preventing and treatingviral diseases, tumors or cancers.

The virus diseases include but are not limited to hepatitis A, hepatitisB, hepatitis C, other types of hepatitis, infections caused byEpstein-Barr virus, Cytomegalovirus, herpes simplex viruses, otherherpes viruses, papovaviruses, poxviruses, picornaviruses, adenoviruses,rihnoviruses, human T cell leukaemia viruses I, human T cell leukaemiaviruses II, or human T cell leukemia viruses III.

Therefore, this invention provides a method for inhibiting virusreplication or virus infected cells by contacting said virus or infectedcells with an effective amount of the super-compound interferon or itsequivalent. This super-compound interferon is useful in preventing ortreating the following cancers or tumors:

Cancer Skin Cancer Basal Cell Carcinoma Malignant Melanoma Renal cellcarcinoma Liver Cancer Thyroid Cancer Rhinopharyngeal Cancer SolidCarcinoma Prostate Cancer Tummy Cancer Esophagus Cancer Recta CancerPancreas Cancer Mammary Cancer Ovarian Cancer & Superficial BladderCancer Hemangioma Epidermoid Carcinoma Cervical Cancer Non-small CellLung Cancer Small Cell Lung Cancer Glioma Malignant Leucocythemia AcuteLeucocythemia Hemal Chronic Leucocythemia Disease Chronic MyelocyticLeukemia Hairy Cell Leukemia Lymphadenoma Multiple Myeloma PolycythemiaVera Others Kaposi's Sarcoma

Accordingly, this invention provides a method for inhibiting tumor orcancer cell growth by contacting the super-compound interferon or itsequivalent with said tumor or cancer cells. In a further embodiment, thesuper-compound interferon inhibits the DNA duplication and secretion ofHBsAg and HBeAg of Hepatitis B Virus.

This invention also provides an artificial gene codes for thesuper-compound interferon or its equivalent. It is within the ordinaryskill to design an artificial gene.

Many methods for generating nucleotide sequence and other molecularbiology techniques have been described previously. See for example,Joseph Sambrook and David W. Russell, Molecular Cloning: A laboratoryManual, December 2000, published by Cold Spring Harbor Laboratory Press.

This invention provides a vector comprising the gene which codes for thesuper-compound interferon or its equivalent.

This invention provides an expression system comprising the vectorcomprising the gene which codes for the super-compound interferon or itsequivalent. The cells include but are not limited to prokaryotic oreukaryotic cells.

This invention also provides a host cell comprising the vectorcomprising the gene which codes for the super-compound interferon or itsequivalent.

This invention provides a process for production of recombinantsuper-compound interferon comprising introducing an artificial gene withselected codon preference into an appropriate host, culturing saidintroduced host in an appropriate condition for the expression of saidcompound interferon and harvesting the expressed compound interferon.

The process may comprise extraction of super-compound interferon fromfermentation broth, collection of inclusion body, denaturation andrenaturation of the harvested protein.

The process may maintain the high efficacy even when the super-compoundinterferon is used with an agent and in a particular concentration. Theprocess also comprises separation and purification of the super-compoundinterferon. The process further comprises lyophilization of the purifiedsuper-compound interferon. The process comprises production of liquidinjection of super-compound interferon.

This invention also provides the produced super-compound interferon bythe above processes.

This invention provides a composition comprising the recombinantsuper-compound interferon or its equivalent and a suitable carrier.

This invention provides a pharmaceutical composition comprising therecombinant super-compound interferon or its equivalent and apharmaceutically acceptable carrier.

This invention provides a method for treating viral diseases or tumor ina subject comprising administering to the subject an effective amount ofthe super-compound interferon or its equivalent.

This invention provides the above-described method wherein the viraldiseases is hepatitis A, hepatitis B, hepatitis C, other types ofhepatitis, infections of viruses caused by Epstein-Barr virus,Cytomegalovirus, herpes simplex viruses, or other type of herpesviruses, papovaviruses, poxviruses, picornaviruses, adenoviruses,rihnoviruses, human T cell leukaemia viruses I, or human T cellleukaemia viruses II, or human T cell leukemia virus III.

This invention provides the above-described method whereinsuper-compound interferon was administered via oral, vein injection,muscle injection, peritoneal injection, subcutaneous injection, nasal,mucosal administration, by inhalation via an inspirator.

This invention provides the above-described method whereinsuper-compound interferon was administered following the protocol ofinjection 9 μg or 15 μg per day, 3 times a week, total 24 weeks.

It was surprising to find that rSIFN-co, the spatial structure of whichhas been changed, is not only a preparation to inhibit the DNAduplication of hepatitis B, but to inhibit the secretion of HBsAg andHBeAg on 2.2.15 cells.

One objective of this invention is to offer a preparation of rSIFN-co todirectly inhibit the DNA duplication of hepatitis B viruses and thesecretion of HBeAg and HBsAg of hepatitis B and decrease them to normallevels.

In one of the results of this invention, rSIFN-co was produced withrecombinant techniques. On the condition of fixed amino acid sequence,the IFN DNA was redesigned according to the E. Coli. codon usage andthen the rSIFN-co gene was artificially synthesized. rSIFN-co cDNA wascloned into the high-expression vector of E. Coli. by DNA recombinanttechniques, and a high expression of rSIFN-co was gained by using ofinduce/activate-mechanism of L-arabinose to activate the transcriptionof P_(BAD) promoter.

Compared with usual thermo-induction, pH induction and IPTG inductionsystems of genetic engineering, arabinose induction/activation systemhas some advantages: (1) Common systems relieve promoter function bycreating a “derepression” pattern. Promoters then induce downstream geneexpression. So temperature and pH change and the addition of IPTG cannotactivate promoters directly. In the system disclosed herein, L-arabinosenot only deactivates and represses but also activates the transcriptionof P_(BAD) promoter which induce a high expression of rSIFN-co.Therefore, the arabinose induction/activation system is a more effectiveexpression system. (2) The relation between Exogenous and L-arabinosedosage is linearity. This means the concentration of arabinose can bechanged to adjust the expression level of the exogenous gene. Therefore,it is easier to control the exogenous gene expression level in E. Coli.by arabinose than by changing temperature and pH value. Thischaracteristic is significant for the formation of inclusion bodies. (3)L-arabinose is resourceful cheap and safe, which, on the contrary, arethe disadvantages of other inducers such as IPTG.

This embodiment creates an effective and resistant rSIFN-co-expressingE. Coli. engineering strain with an L-arabinose induction/activationsystem. The strain is cultivated and fermented under suitable conditionsto harvest the bacterial bodies. Inclusion bodies are then purifiedafter destroying bacteria and washing repeatedly. The end result, massof high-purity, spatial-configuration-changed rSIFN-co protein for thisinvention and for clinical treatment, was gained from denaturation andrenaturation of inclusion bodies and a series of purification steps.

The following are some rSIFN-co preparations: tablets, capsules, oralliquids, pastes, injections, sprays, suppositories, and solutions.Injections are recommended. It is common to subcutaneously inject orvein-inject the medicine. The medicine carrier could be any acceptancemedicine carrier, including carbohydrate, cellulosum, adhesive,collapse, emollient, filling, add-dissolve agent, amortization,preservative, add-thick agent, matching, etc.

This invention also provides a pharmaceutical composition comprising theabove composition and a pharmaceutically acceptable carrier.

For the purposes of this invention, “pharmaceutically acceptablecarriers” means any of the standard pharmaceutical carriers. Examples ofsuitable carriers are well known in the art and may include, but are notlimited to, any of the standard pharmaceutical carriers such as aphosphate buffered saline solution and various wetting agents. Othercarriers may include additives used in tablets, granules and capsules,etc. Typically such carriers contain excipients such as starch, milk,sugar, certain types of clay, gelatin, stearic acid or salts thereof,magnesium or calcium stearate, talc, vegetable fats or oils, gum,glycols or other known excipients. Such carriers may also include flavorand color additives or other ingredients. Compositions comprising suchcarriers are formulated by well-known conventional methods.

This invention will be better understood from the examples which follow.However, one skilled in the art will readily appreciate that thespecific methods and results discussed are merely illustrative of theinvention as described more fully in the claims which follow thereafter.

EXPERIMENTAL DETAILS Example 1

rSIFN-co is a new interferon molecule constructed according toconservative amino acid in human IFN-α subtype with genetic engineeringmethod. It has been proven that rSIFN-co has broad-spectrum IFNactivity, such as high antivirus and tumor inhibition activity,especially for effectively treating hepatitis C.

E. Coli. codon was used to redesign rSIFN-co cDNA and then artificiallysynthesize cDNA of rSIFN-co from published rSIFN-co DNA sequences anddeduced amino acid sequences (FIG. 1).

In order to get pure rSIFN-co protein, rSIFN-co cDNA was cloned into E.Coli. high-expression vector, and L-arabinose, which can activate strongP_(BAD) promoter in vectors, was used to induce high expression ofrSlFN-co gene.

Synthesis of E. Coli. cDNA SequenceRedesign of rSIFN-co cDNA Sequence

rSIFN-co cDNA was redesigned according to the codon usage of E. Coli. toachieve high expression in E. Coli. Deduced amino acid sequence from theredesigned cDNA sequence of rSIFN-co is completely coincidental withprimitive amino acid sequence of published rSIFN-co (FIG. 1).

rSIFN-co cDNA Sequence SynthesisrSIFN-co cDNA 5′-Terminus and 3′-Terminus Semi-Molecular Synthesis

Two semi-moleculars can be directly synthesized: rSIFN-co cDNA5′-terminus 280 bp (fragment I) and 3′-terminus 268 bp (fragment II) byPCR. There are 41 bp overlapping among fragment II and fragment I.

(1) Chemical Synthesis Oligodeoxynucleotide Fragment:

Oligomer A: 5′ATGTGCGACCTGCCGCAGACCCACTCCCTGGGTAACCGTCGTGCTCTGATCCTGCTGGCTCAGATGCGTCGTATCTCCCCGTTCTCCTGCCTGA AAGACCGTCACGAC3′Oligomer B: 5′CTGAAAGACCGTCACGACTTCGGTTTCCCGCAGGAGAGGTTCGACGGTAACCAGTTCCAGAAGCTCAGGCTATCTCCGTTCTGCACGAAATGAT CCAGCAGACCTTC3′Oligomer C: 5′GCTGCTGGTACAGTTCGGTGTAGAATTTTTCCAGCAGGGATTCGTCCCAAGCAGCGGAGGAGTCTTTGGTGGAGAACAGGTTGAAGGTCTGCTG GATCATTTC3′ Oligomer D:5′ATCCCTGCTGGAAAAATTCTACACCGAACTGTACCAGCAGCTGAACGACCTGGAAGCTTGCGTTATCCAGGAAGTTGGTGTTGAAGAAACCCCG CTGATGAAC3′ Oligomer E:5′GAAGAAACCCCGCTGATGAACGTTGACTCCATCCTGGCTGTTAAAAAATACTTCCAGCGTATCACCCTGTACCTGACCGAAAAAAAATACTCCC CGTGCGCTTGGG3′Oligomer F: 5′TTATTCTTTACGACGCAGACGTTCCTGCAGGTTGGTGGACAGGGAGAAGGAACGCATGATTTCAGCACGAACAACTTCCCAAGCGCACGGGGAG TATTTTTTTTCGGTCAGG3′PCR I for Fragment I: oligodeoxynucleotide B as template,oligodeoxynucleotide A and C as primers, synthesized 280 bp Fragment I.

PCR I mixture (units: μl) sterilized distilled water 39 10 × Pfu buffer(Stratagen American Ltd.) 5 dNTP mixture (dNTP concentration 2.5 mmol/L)2 Oligomer A primer (25 μmol/L) 1 Oligomer C primer (25 μmol/L) 1Oligomer B template (1 μmol/L) 1 Pfu DNA polymerase (Stratagen AmericanLtd.) 1 (25 U/μl) Total volume 50 μl PCR cycle: 95 I 2 m→(95° C. 45s→65° C. 1 m→72° C. 1 m) × 25 cycle→72° C. 10 m→ ′4° C.

PCR II for Fragment II: oligodeoxynucleotide E as template,oligodeoxynucleotide D and F as primers, synthesized 268 bp Fragment II.

PCR II mixture (units: μl) sterilized distilled water 39 10 × Pfu buffer(Stratagen American Ltd.) 5 dNTP mixture (dNTP concentration 2.5 mmol/L)2 Oligomer D primer (25 μmol/L) 1 Oligomer F primer (25 μmol/L) 1Oligomer E template (1 μmol/L) 1 Pfu DNA polymerase (Stratagen AmericanLtd.) 1 (25 U/μl) Total volume 50 μl PCR cycle: the same as PCR IAssembling of rSIFN-co cDNA

Fragment I and II were assembled together to get the complete cDNAmolecular sequence of rSIFN-co using the overlapping and extending PCRmethod. Restriction enzyme Nde I and Pst I were introduced to clonerSIFN-co cDNA sequence into plasmid.

(1) Chemical Synthesis Primers

Oligomer G: 5′ATCGGCCATATGTGCGACCTGCCGCAGACCC3′ Oligomer H:5′ACTGCCAGGCTGCAGTTATTCTTTACGACGCAGACGTTCC3′

(2) Overlapping and Extending PCR

PCR mixture (units: μl) sterilized distilled water 38 10 × Pfu buffer(Stratagen American Ltd.) 5 dNTP mixture (dNTP concentration 2.5 mmol/L)2 primer G (25 μmol/L) 1 primer H (25 μmol/L) 1 *fragment I preduction(1 μmol/L) 1 *fragment II preduction (1 μmol/L) 1 Pfu DNA polymerase(Stratagen American Ltd.) 1 (2.5 U/μl) Total volume 50μ *Separate andpurify PCR production with StrataPrep PCR purification kit produced byStratagen American Ltd. And dissolve into sterilized distilled water.PCR cycle: the same as PCR IrSIFN-co Gene Clone and Sequence Analysis

pLac T7 plasmid as cloning vector. pLac T7 plasmid is reconstructed withpBluescript II KS(+) plasmid produced by Stratagen (FIG. 3).

Purified PCR production of rSIFN-co cDNA with StrataPrep PCRpurification kit. Digest cDNA and pLac T7 plasmid with NdeI and PstI.Run 1% agarose gel electrophoresis and separate these double-digestedDNA fragments. Recover 507 bp long rSIFN-co DNA fragment and 2.9 kbplasmid DNA fragment. Ligate these fragments by T4 DNA ligase to form arecombinant plasmid. Transform DH_(5α) competent cells (Gibco) with therecombinant plasmid, culture at 37° C. overnight. Identify the positiverecombinant colony, named pHY-1.

Run DNA sequencing with SequiTherm™ Cycle Sequencing Kit produced byAmerican Epicentre Technologies Ltd using L1-COR Model 4000L. Primersare T7 and T3 common sequence primer, the DNA sequencing result matchestheoretic design.

Purify the rSIFN-co, sequence the N-terminus amino acids, the N-terminusamino acid sequence matches experimental design which is as follows:

N-Cys-Asp-Leu-Pro-Gln-Thr-His-Ser-Leu-Gly- Asn-Arg-Arg-Ala-Leu-

Construction, Transformation, Identification, and Hereditary Stabilityof Expression Vector Construction and Transformation of ExpressionVector

Digested E. Coli. expression vector pHY-4 (see FIG. 3) with Nde I tolinearize and subsequently digest with Xba I. Run 1% agarose gelelectrophoresis, and purify the 4.8 kb pHY-4 Nde I-Xba I digest fragmentwith QIAEX II kit produced by QIAGEN Germany Ltd.

At the same time, the pHY-4 plasmid is double digested with Nde I-Xba I.Run 1% agarose gel electrophoresis and purify the 715 bp fragment.Ligate the rSIFN-co and pHY-4 fragments with T4 DNA ligase to constructthe recombinant plasmid (See FIG. 4). Transform DH_(5α) competent cellswith the recombinant plasmid. Spread the transformed cells on LB platewith Amp, 37° C. culture overnight.

Positive Cloning Strain Screening

Randomly choose E. Coli. colonies from above LB-plate, screening thepositive strains containing recombinant vector by endonuclease digestingand PCR analysis. Name one of the positive recombinant plasmid pHY-5,and name the strain containing pHY-5 plasmid PVIII. Amplify and storethe positive strain with glycerol in −80° C.

High Expression of rSIFN-co Gene in E. Coli.

In pHY-5 plasmid, rSIFN-co gene is under control of strong promoterP_(BAD). This promoter is positively and negatively regulated by theproduct of the gene araC. AraC is a transcriptional regulator that formsa complex with arabinose. In the absence of arabinose, the AraC dimerbinds O₂ and I₁ forming a 210 bp loop. This conformation leads to acomplete inhibition of transcription. In the presence of arabinose, thedimer is released from O₂ and binds I₁ and I₂ leading to transcription.Arabinose binding deactivates, represses and even activates thetranscription of P_(BAD) promoter, which stimulates P_(BAD) inducinghigh expression of rSIFN-co. rSIFN-co expression level in PVIII is morethan 50% of the total E. Coli. protein.

SUMMARY

RSIFN-CO is a new interferon molecule artificially built according tothe conservative amino acid of human α interferons. It has been provenas a effective anti-hepatitis drug. In order to get enough pure rSIFN-coprotein, a stable recombinant E. Coli. strain which high expressesrSIFN-co protein was constructed.

First, according to published rSIFN-co amino acid sequence, E. Coli.codon was used to synthesize whole cDNA of rSIFN-co. This DNA fragmentwas sequenced and proved that the 501 bp codon sequence and TAAtermination codon sequence are valid and identical with theocraticdesign. Subsequent analysis revealed that the N-terminus amino acidsequence and amino acid composed of rSIFN-co produced by the recombinantstrain were both identical to the prediction.

The rSIFN-co cDNA was cloned into E. Coli. high-expression vector pHY-4plasmid to construct the recombinant plasmid pHY-5. E. Coli. LMG194strain was further transformed with pHY-4 plasmid to get stable rSIFN-cohigh-expression transformant. This transformant was cultured for 30generations. The heredity of pHY-5 recombinant plasmid in E. Coli.LMG194 was normal and stable, and the expression of rSIFN-co was highand steady.

E. Coli. LMG194, which contains recombinant pHY-5 plasmid, is actuallyan ideal high-expression engineering strain.

REFERENCES

-   1. Blatt L M, Davis J M, Klein S B. et al. The biologic activity and    molecular characterization of a novel synthetic interferon-alpha    species, consensus interferon. Journal of Interferon and Cytokine    Research, 1996; 16(7):489-499.-   2. Alton, K. et al: Production characterization and biological    effects of recombinant DNA derived human IFN-α and IFN-γ analogs.    In: De Maeger E, Schellekens H. eds. The Biology of Interferon    System. 2nd ed. Amsterdam: Elsevier Science Publishers, 1983:    119-128-   3. Pfeffer L M. Biologic activity of natural and synthetic type 1    interferons. Seminars in Oncology, 1997; 24 (3 suppl 9):S9-63-S9-69.-   4. Ozes O N, Reiter Z, Klein S, et al. A comparison of    interferon-con1 with natural recombinant interferons-(: antiviral,    antiproliferative, and natural killer-inducing activities. J.    Interferon Res., 1992; 12:55-59.-   5. Heathcote E J L, Keeffe E B, Lee S S, et al. Re-treatment of    chronic hepatitis C with consensus interferon. Hepatology, 1998;    27(4):1136-1143.-   6. Klein M L, Bartley T D, Lai P H, et al. Structural    characterization of recombinant consensus interferon-alpha. Journal    of Chromatography, 1988; 454:205-215.-   7. The Wisconsin Package, by Genetics Computer Group, Inc. Copyright    1992, Medison, Wis., USA-   8. Nishimura, A et al: A rapid and highly efficient method for    preparation of competent E. coli cells. Nuclei. Acids Res. 1990,    18:6169-   9. All molecular cloning techniques used are from: Sambrook,    J., E. F. Fritsch and T. Maniatis. Molecular Cloning: A laboratory    manual, 2nd ed. CSH Laboratory Press, Cold Spring Harbour, N.Y.    1989.-   10. Guzman, L. M et al: Tight regulation, modulation, and high-level    express-ion by vectors containing the arabinose PBAD promoter. J.    Bacteriol. 1995, 177: 4121˜4130.    rSIFN-co cDNA Sequence Designed According to E. Coli. Codon Usage    and Deduced rSIFN-co Amino Acid Sequence

   5'           11         21         31        41          51+1   M   C  D   L  P  Q  T   H  S  L   G  N  R   R  A  L  I   L  L  A   1 ATGTGCGACC TGCCGCAGAC CCACTCCCTG GGTAACCGTC GTGCTCTGAT CCTGCTGGCT     TACACGCTGG ACGGCGTCTG GGTGAGGGAC CCATTGGCAG CACGAGACTA GGACGACCGA     5'           71          81        91        101         111+1   Q  M   R  R  I  S  P   F  S  C   L  K  D   R  H  D  F    G  F  P  61 CAGATGCGTC GTATCTCCCC GTTCTCCTGC CTGAAAGACC GTCACGACTT CGGTTTCCCG     GTCTACGCAG CATAGAGGGG CAAGAGGACG GACTTTCTGG CAGTGCTGAA GCCAAAGGGC    5'            131       141       151        161        171+1     Q  E  E   F  D  G  N   Q  F  Q   K  A  Q   A  I  S  V   L  H  E 121 CAGGAAGAAT TCGACGGTAA CCAGTTCCAG AAAGCTCAGG CTATCTCCGT TCTGCACGAA     GTCCTTCTTA AGCTGCCATT GGTCAAGGTC TTTCGAGTCC GATAGAGGCA AGACGTGCTT    5'           191       201         211        221       231+1    M  I  Q  Q  T  F  N   L  F  S   T  K  D   S  S  A  A   W  D  E 181 ATGATCCAGC AGACCTTCAA CCTGTTCTCC ACCAAAGACT CCTCCGCTGC TTGGGACGAA     TACTAGGTCG TCTGGAAGTT GGACAAGAGG TGGTTTCTGA GGAGGCGACG AACCCTGCTT    5'           251       261         271       281        291+1    S  L  L  E  K  F  Y   T  E  L   Y  Q  Q   L  N  D  L   E  A  C 241 TCCCTGCTGG AAAAATTCTA CACCGAACTG TACCAGCAGC TGAACGACCT GGAAGCTTGC     AGGGACGACC TTTTTAAGAT GTGGCTTGAC ATGGTCGTCG ACTTGCTGGA CCTTCGAACG    5'            311        321       331        341        351+1   V  I  Q     E V G V    E E  T P   L  M  N    V  D  S    I  L  A 301 GTTATCCAGG AAGTTGGTGT TGAAGAAACC CCGCTGATGA ACGTTGACTC CATCCTGGCT     CAATAGGTCC TTCAACCACA ACTTCTTTGG GGCGACTACT TGCAACTGAG GTAGGACCGA    5'          371        381         391       401        411+1   V  K  K  Y  F   Q  R   I  T  L   Y  L  T   E  K   K   Y  S  P  C 361 GTTAAAAAAT ACTTCCAGCG TATCACCCTG TACCTGACCG AAAAAAAATA CTCCCCGTGC     CAATTTTTTA TGAAGGTCGC ATAGTGGGAC ATGGACTGGC TTTTTTTTAT GAGGGGCACG    5' 431 441 451 461 471+1   A  W  E  V   V  R  A   E  I  M    R  S  F   S   L  S   T  N  L  Q 421 GCTTGGGAAG TTGTTCGTGC TGAAATCATG CGTTCCTTCT CCCTGTCCAC CAACCTGCAG     CGAACCCTTC AACAAGCACG ACTTTAGTAC GCAAGGAAGA GGGACAGGTG GTTGGACGTC    5'           491        501 +1   E  R  L    R  R  K  E    # 481 GAACGTCTGC GTCGTAAAGA ATAA      CTTGCAGACG CAGCATTTCT TATT

Example 2 Separation and Purification of rSIFN-co 1. Fermentation

Inoculate the recombinant strain in LB media, shaking (200 rpm) under37° C. overnight (approximate 18 h), then add 30% glycerol to thefermentation broth to get final concentration of 15%, allotted to 1 mltube and kept in −20° C. as seed for production.

Add 1% of the seed to LB media, shaking (200 rpm) under 37° C. overnightto enlarge the scale of the seed, then add to RM media with a ratio of10%, culturing under 37° C. Add arabinose (20% solution) to 0.02% as aninductor when the OD600 reaches about 2.0. 4 hours after that, stop theculture process, collect the bacteria by centrifuge, resuspend thepellet with buffer A, and keep in −20° C. overnight. Thaw and break thebacteria by homogenizer, then centrifuge. Wash the pellet with buffer B,buffer C, and distilled water to get a relatively pure inclusion body.

2. Denaturation and Renaturation

Dissolve the inclusion body in Guanidine-HCl (or urea) of 6 mol/L. Thesolution will be a little cloudy. Centrifuge it at a speed of 10000 rpm.Determine the protein concentration of the supernatant. This supernatantis called “denaturation solution.” Add the denaturation solution torenaturation buffer, and keep the final protein concentration under 0.3mg/ml. It is better to add the totally denaturation solution in threesteps instead of one step. Keep the solution overnight under 4° C.Afterwards, dialyze 10 mol/L, 5 mol/L PB buffer and distilled water,then adjust its pH by 2 mol/L HAc-NaAc. Let it stand, then filtrate.

3. Purification

POROS HS/M Anion Exchange Chromatography:

Chelating Sepharose™ fast flow: Add PB buffer of 0.2 mol/L (pH 6.6) andNaCl of 4 mol/L in the solution from HS to adjust solution pH to pH 6.0and NaCl concentration to 1 mol/L.

Condense the eluted solution by POROS HS/M. Sometimes a purification bysephacryl S-100 step can be added to meet stricter purity requirements.

Note:

-   Buffer A: 100 mmol/L Tris-HCl, pH 7.5-10 mmol/L EDTA-100 mmol/L NaCl-   Buffer B: 50 mmol/L Tris-HCl, pH 7.5-1 mol/L Urea-10 mmol/L    EDTA-0.5% Triton X-100-   Buffer C: 50 mmol/L Tris-HCl, pH 7.5-2 mol/L Urea-10 mmol/L    EDTA-0.5% Triton X-100-   Buffer D: 1 mol/L NaCl—50 mmol/L Na₂HPO₄ (pH 5.5)-   Buffer E: 1 mol/L NaCl—50 mmol/L Na₂HPO₄ (pH 5.0)-   Buffer F: 1 mol/L NaCl—50 mmol/L Na₂HPO₄ (pH 4.0)-   Buffer G: 1 mol/L NaCl—50 mmol/L Na₂HPO₄ (pH 3.6)

Renaturation buffer: 0.5 mol/L Arginine-150 mmol/L Tris-HCl, pH 7.5-0.2mmol/L EDTA

LB Media: 1 L Tryptone 10 g Yeast extracts 5 g NaCl 10 g

RM Media: 1 L Casein 20 g MgCl 1 mmol/L (0.203 g) Na₂HPO₄ 4 g; KH₂PO₄ 3g, NaCl 0.5 g NH₄Cl 1 g

After purification, the buffer was changed to PBS (pH 7.0) along withthe step of condensing by POROS HS/M. This is called the “Protein StockSolution.” It can directly used in the preparation of injections orsprays, or stored at 2-8° C.

Formula for Injection:

Solution Lyophilized powder Solution of rSIFN- 34.5 μg/ml 34.5 μg/ml coPB (pH 7.0) 25 mmol/L 10 mmol/L Glycine — 0.4 mol/L NaCl 0.1 mol/L —

For Spray:

EDTA 0.01% Tween 80 0.05% Trisodium citrate 10 mmol/L Glycerol 1.26%Sodium Chloride 0.03% Phenylmethanol 0.5% HSA 0.1% rSIFN-co 10 μg/ml

Quality Control Process

During purification tests for protein content, protein purity, specificactivity and pyrogen are conducted after each step. When the stocksolution is obtained, all the tests listed in the table are done oneafter the other.

The quality of the product is controlled according to “ChineseRequirements for Biologics”

1. Original Protein Solution

Lowry Item of Test Method Protein Stock Solution: Test for ProteinContent Lowry Test for Protein Purity Non-reductive SDS-PAGE (sodiumdodecyl sulfate polyacrylamide gel electrophoresis) HPLC Analysis Testfor Molecular Weights Reductive SDS-PAGE Test for Specific ActivityAccording to Method in “Specific Activity Test of Interferon Test forLeftover Exogenetic Using DNA Labeling and DNA Detection Kit Test forActivity of According to Method in Leftover Antibiotics “Chemical andOther Test Methods for Biologics” Test for Bacterial Endotoxin Accordingto Method in “Requirements for Bacterial Endotoxin Test of Biologics”Test for Isoelectronic Point Isoelectric Focusing Electrophoresis Testfor Identify UV spectrum (range of Characteristics of the wavelength:190-380 nm) Protein Peptide Mapping (hydrolyzed by pancreatic enzyme,analyzed by C-18 column) N-terminal Sequence Test C-terminal SequenceTest Circular Dichroism Amino Acid Analysis Semi-finished Product Testfor Bacterial Endotoxin According to Method in “Requirements forBacterial Endotoxin Test of Biologics” Product Appearance Check ChemicalAccording to Method in “Chemical and Other Test Methods for Biologics”Test for Specific Activity According to Method in “Specific ActivityTest of Interferon Sterility Test According to Method in “c” AbnormalToxicity Test Test on Mouse Pyrogen Test According to Method in“Requirements for Pyrogen Test of Biologics” Test for Stability ofProduct Note: “Chemical and Other Test Methods for Biologics”,“Requirements for Pyrogen Test of Biologics” and “Requirements forBacterial Endotoxin Test of Biologics” all can be found in the “ChineseRequirements for Biologics.” “Chinese Requirements for Biologics,″ PANZhengan, ZHANG Xinhui, DUAN Zhibing, et al. Chinese BiologicsStandardization committee. Published by Chemical Industry PublishingCompany, 2000.

Example 3 Stability of lyophilized Powder of Recombinant Super-CompoundInterferon Injection

The stability experiments were carried out with samples of lyophilizedpowder of recombinant super-compound interferon (rSIFN-co) injection intwo specifications and three batches. The experiments started on April,2000.

1. Sample Source

Samples were supplied by Sichuan Huiyang Life-engineering Ltd., SichuanProvince. Lot: 990101-03, 990101-05, 990102-03, 990102-05, 990103-03,990103-05

2. Sample Specifications

Every sample in this experiment should conform with the requirements inthe table below.

TABLE 1 Standard of Samples in Experiment Items Standards 1. Appearancewhite loose powder 2. Dissolving dissolve rapidly in injectionwater(within time 2 min) at room temperature 3. Clarity colorless liquidor with little milk-like glisten; should not be cloudy, impurity or withindiscernible deposit 4. pH value 6.5~7.5 5. Potency 80%~150% ofindicated quantity (9 μg: 4.5 × (IU/dose) 10⁶ IU, 15 μg: 7.5 × 10⁶ IU)6. Moisture no more than 3.0% (W/W)

3. Experiment Content

15.3.1 Test samples at 2˜8° C.: The test samples were put into a 2-8° C.refrigerator, then the above items of these samples were respectivelytested in the 1^(st), 3^(rd), 6^(th), 9^(th), 12^(th), 18^(th), 24^(th),30^(th), 36^(th) month. The results were recorded.

15.3.2 Test samples at 25° C.: The test samples were put into athermostat at 25° C., then the above items of these samples wererespectively tested in the 1^(st), 3^(rd), 6^(th), 9^(th), 12^(th),18^(th), 24^(th), 30^(th) month. The results were recorded.

15.3.3 Test samples at 37° C.: The test samples were put into athermostat at 37° C., then the above items of these samples wererespectively tested in the 1^(st), 3^(rd), 6^(th), 9^(th), 12^(th),18^(th), 24^(th) month. The results were recorded.

4. Results and Conclusion

1) At 37° C., according to data collected at designated points duringtesting and compared with data before testing, the potency begandescending from the 6^(th) month and the changes in the three batcheswere similar. The appearance of other items had no changes.

2) At 25° C., according to data collected at designated points duringtesting and compared with data before the testing, the potency only hada little change, and the changes in the three batches were similar. Theappearance of other items had no changes.

3). At 2-8° C., according to data collected at designated points duringtesting and compared with data before testing, the potency of the threebatches all were stable. The appearance of other items also had nochanges.

In conclusion, it is suggested that the lyophilized powder ofrecombinant super-compound interferon for injection should be betterstored and transported at low temperatures. Without such conditions, theproduct can also be stored for short periods (i.e. 3 months) at roomtemperature.

Example 4 rSIFN-co Inhibits HBV-DNA Duplication and Secretion of HBsAgand HBeAg Materials

Solvent and Dispensing Method: Add 1 ml saline into each vial, dissolve,and mix with MEM culture medium at different concentrations. Mix on thespot.

Control drugs: IFN-α2b (Intron A) as lyophilized powder, purchased fromSchering Plough. 3×10⁶ U each, mix to 3×10⁶ IU/ml with culture medium;Infergen® (liquid solution), purchased from Amgen, gig, 0.3 ml each,equal to 9×10⁶ IU, and mix with 9×10⁶ IU/ml culture medium preserve at4° C.; 2.2.15 cell: 2.2.15 cell line of hepatoma (Hep G2) cloned andtransfected by HBV DNA, constructed by Mount Sinai Medical Center.

Reagent: MEM powder, Gibco American Ltd. cattle fetal blood serum,HycloneLab American Ltd. G-418 (Geneticin); MEM dispensing, GibcoAmerican Ltd.; L-Glutamyl, imported and packaged by JING KE ChemicalLtd.; HBsAg and HBeAg solid-phase radioimmunoassay box, NorthwardReagent Institute of Chinese Isotope Ltd.; Biograncetina, Northern ChinaMedicine; And Lipofectin, Gibco American Ltd.

Experimental goods and equipment: culture bottle, Denmark Tunclon™;24-well and 96-well culture board, Corning American Ltd.; Carbon Dioxidehatching box, Shel-Lab American Ltd.; MEM culture medium 100 ml:10%cattle fetal blood serum, 3% Glutamyl 1%, G418 380 μg/ml, biograncetina50 U/ml.

Method:

2.2.15 cell culture: Added 0.25% pancreatic enzyme into culture box withfull of 2.2.15 cell, digest at 37° C. for 3 minutes, and add culturemedium to stop digest and disturb it to disperse the cells, reproducewith ratio of 1:3. They will reach full growth in 10 days.

Toxicity test: Set groups of different concentrations and a controlgroup in which cell is not acted on with medicine. Digest cell, anddispense to a 100,000 cell/ml solution. Inoculate to 96-well cultureboard, 200 μl each well, culture at 37° C. for 24 h with 5% CO₂. Testwhen simple cell layer grows.

Dispense rSIFN-co to 1.8×10⁷¹ U/ml solution than prepare a series ofsolutions diluted at two-fold gradients. Add into 96-well culture board,3 wells per concentration. Change the solution every 4 days. Testcytopathic effect by microscope after 8 days. Fully destroy as 4, 75% as3, 50% as 2, 25% as 1, zero as O. Calculate average cell lesion andinhibition rate of different concentrations. Calculate TC50 and TC0according to the Reed Muench method.

${{TC}\; 50} = {{Antilog}\left( {B + {\frac{50 - B}{A - B} \times C}} \right)}$

A=log>50% medicine concentration, B=log<50% medicine concentration,C=log dilution power

Inhibition test for HBeAg and HBsAg: Separate into positive and negativeHBeAg and HBsAg contrast groups, cell contrast group and medicineconcentration groups. Inoculate 700,000 cells/ml of 2.2.15 cell into6-well culture board, 3 ml each well, culture at 37° C. for 24 h with 5%CO₂, then prepare 5 gradiently diluted solutions with 3-fold as thegrade (Prepare 5 solutions, each with a different protein concentration.The concentration of Solution 2 is 3 times lower than that of Solution1, the concentration of Solution 3 is 3 times lower than that ofSolution 2, etc.) 4.5×10⁶ IU/ml, 1.5×10⁶ IU/ml, 0.5×10⁶ IU/ml, 0.17×10⁶1U/ml, and 0.056×10⁶ IU/ml, 1 well per concentration, culture at 37° C.for 24 h with 5% CO₂. Change solutions every 4 days using the samesolution. Collect all culture medium on the 8^(th) day. Preserve at −20°C. Repeat test 3 times to estimate HBsAg and HBeAg with solid-phaseradioimmunoassay box (Northward Reagent Institute of Chinese IsotopeLtd.). Estimate cpm value of each well with a γ-accounting machine.

Effects calculation: Calculate cpm mean value of contrast groups anddifferent-concentration groups and their standard deviation, P/N valuesuch as inhibition rate, IC50 and SI.

-   -   1)

${{Antigen}\mspace{14mu} {inhibition}\mspace{14mu} {rate}\mspace{14mu} (\%)} = {\frac{A - B}{A} \times 100}$

-   -    A=cpm of control group; B=cpm of test group;    -   2) Counting the half-efficiency concentration of the medicine

${{Antigen}\mspace{14mu} {inhibition}\mspace{14mu} {IC}\; 50} = {{Antilog}\left( {B + {\frac{50 - B}{A - B} \times C}} \right)}$

A=log>50% medicine concentration, B=log<50% medicine concentration,C=log dilution power

-   -   3) SI of interspace-conformation changed rSIFN-co effect on        HBsAg and HBeAg in 2.2.15 cell culture:

${SI} = \frac{{TC}\; 50}{{IC}\; 50}$

-   -   4) Estimate the differences in cpm of each dilution degree from        the control group using student t test

Southern blot: (1) HBV-DNA extract in 2.2.15 cell: Culture cell 8 days.Exsuction culture medium (Separate cells from culture medium by means ofdraining the culture medium.). Add lysis buffer to break cells, thenextract 2 times with a mixture of phenol, chloroform and isoamyl alcohol(1:1:1), 10,000 g centrifuge. Collect the supernatant adding anhydrousalcohol to deposit nucleic acid. Vacuum draw, re-dissolve into 20 μl TEbuffer. (2) Electrophoresis: Add 6×DNA loading buffer, electrophoresison 1.5% agarose gel, IV/cm, at fixed pressure for 14-18 h. (3)Denaturation and hybridization: respectively dip gel into HCl,denaturation buffer and neutralization buffer. (4) Transmembrane: Makean orderly transfer of DNA to Hybond-N membrane. Bake, hybridize andexpose with dot blot hybridization. Scan and analyze relative densitywith gel-pro software. Calculate inhibition rate and IC50.

Results

Results from Tables 1, 2 and 3 show: After maximum innocuousconcentration exponent culturing for 8 days with 2.2.15 cell, the maximais 9.0±0×10⁶ IU/ml average inhibition rate of maximum innocuousconcentration rSIFN-co to HBeAg is 46.0±5.25% (P<0.001), IC50 is4.54±1.32×10⁶ IU/ml, SI is 3.96; rate to HBsAg is 44.8±6.6%, IC50 is6.49±0.42×10⁶ IU/ml, SI is 2.77. This shows that rSIFN-co cansignificantly inhibit the activity of HBeAg and HBsAg, but that the IFNof the contrast group and Infergen® cannot. It has also been proved inclinic that rSIFN-co can decrease HBeAg and HBsAg or return them tonormal levels.

TABLE 1 Results of inhibition rate of rSIFN-co to HBsAg and HBeAgInhibition rate Average Concentration First Second Third First SecondThird inhibition Accumu- (×10⁴IU/ml) well well well well well rate ratelation First batch: (rSIFN-co) Inhibition effect to HBeAg 900 9026 897610476 0.436227 0.43935 0.345659 0.407079 0.945909 300 9616 12082 100980.3993754 0.245347 0.369269 0.337997 0.5388299 100 9822 16002 128000.386508 0.0005 0.2005 0.195836 0.200833 33.33333 15770 19306 168240.014991 0 0 0.004997 0.0049969 11.11111 19172 22270 18934 0 0 0 0 0Control Cell 16010 Blank 0 Dilution 3 Inhibition effect to HBsAg 9007706 7240 7114 0.342155 0.381936 0.392693 0.372261 0.922258 300 88567778 9476 0.2439816 0.336008 0.191053 0.257014 0.5499972 100 10818 1072010330 0.07649 0.084856 0.118149 0.093165 0.292983 33.33333 10744 1111410570 0.082807 0.051221 0.097661 0.07723 0.1998179 11.11111 10672 935210810 0.088953 0.201639 0.077173 0.122588 0.122588 Control Cell 11714Blank 0 Dilution 3 Second batch: (rSIFN-co) Inhibition effect to HBeAg900 7818 8516 9350 0.554378 0.514592 0.467054 0.512008 1.371181 30010344 10628 9160 0.4103967 0.394209 0.477884 0.427497 0.8591731 10012296 14228 13262 0.299134 0.18901 0.244072 0.244072 0.4316522 33.3333315364 17414 16188 0.124259 0.00741 0.77291 0.069653 0.1876045 11.1111117386 13632 15406 0.009006 0.222982 0.121865 0.117951 0.117951 ControlCell 16962 Blank 0 Dilution 3 Inhibition effect to HBsAg 900 5784 61985792 0.498265 0.462353 0.497571 0.486063 0.893477 300 7150 8534 83180.379771 0.259715 0.278452 0.30598 0.4074138 100 9830 11212 102100.147294 0.027412 0.11433 0.096345 0.101434 33.33333 13942 12368 13478 00 0 0 0.0050891 11.11111 12418 11634 11352 0 0 0.015267 0.0050890.005089 Control Cell Blank 0 Dilution 3 Third batch: (rSIFN-co)Inhibition effect to HBeAg 900 9702 9614 8110 0.428016 0.433204 0.5218720.461031 1.316983 300 8914 10032 8870 0.4744723 0.40856 0.4770660.453366 0.8559525 100 16312 12688 13934 0.038321 0.251975 0.1785170.156271 0.402586 33.33333 15080 12814 13288 0.110954 0.244547 0.2166020.190701 0.2463153 11.11111 21928 15366 15728 0 0.094093 0.0727510.0055615 0.055615 Control Cell 17544 Blank 0 Dilution 3 Inhibitioneffect to HBsAg 900 5616 6228 5346 0.496864 0.442035 0.521054 0.4866510.763125 300 8542 8590 7096 0.234725 0.230425 0.364272 0.2764740.2764738 100 11420 11360 11394 0 0 0 0 0 33.33333 12656 11582 13110 0 00 0 0 11.11111 13142 12336 13342 0 0 0 0 0 Control Cell 11528 Blank 0Dilution 3 Results of inhibition rate of rSIFN-co to HBsAg and HBeAgAccumulated Concentration inhibition (×10⁴IU/ml) 1-Accumulation rateFirst batch: (rSIFN0-co) Inhibition effect to HBeAg 900 0.5929210.614693546 300 1.254924 0.300392321 100 2.059088 0.08867188 33.333333.054091 0.001633453 11.11111 4.054091 0 Control IC50 602.74446016Inhibition effect to HBsAg 900 0.627739 0.595006426 300 1.3707240.286349225 100 2.27756 0.113977019 33.33333 3.20033 0.05876740811.11111 4.077742 0.02918541 Control IC50 641.7736749 Second batch:(rSIFN-co) Inhibition effect to HBeAg 900 0.487992 0.737521972 3001.060496 0.447563245 100 1.816423 0.19201839 33.33333 2.746770.063933386 11.11111 3.628819 0.03148073 Control IC50 365.9357846Inhibition effect to HBsAg 900 0.513937 0.634835847 300 1.2079570.252210647 100 2.111612 0.04583464 33.33333 3.111612 0.00163283511.11111 4.106523 0.001237728 Control IC50 611.0919568 Third batch:(rSIFN-co) Inhibition effect to HBeAg 900 0.538969 0.709599543 3001.085603 0.440859127 100 1.929332 0.172641621 33.33333 2.7386310.082519158 11.11111 3.683017 0.014875633 Control IC50 382.0496935Inhibition effect to HBsAg 900 0.513349 0.597838293 300 1.2368750.182690031 100 2.236875 0 33.33333 0 11.11111 4.236875 0 Control IC50694.7027149 HBeAg: Average IC50: 450.2434 SD: 132.315479 HBsAg: AverageIC50: 649.1894 SD: 42.29580

TABLE 2 Results of inhibition rate of Intron A (IFN-α2b) to HBsAg andHBeAg Inhibition rate Average Accumulated Concentration First SecondThird First Second Third inhibition inhibition (×10⁴IU/ml) well wellwell well well well rate Accumulation 1-Accumulation rate Inhibitioneffect to HBeAg 300 14918 11724 9950 0 0.029711 0.176529 0.0687470.068747 0.931253 0.068746724 100 14868 16890 15182 0 0 0 0 0 1.931253 033.33333 16760 21716 16400 0 0 0 0 0 2.931253 0 11.11111 20854 1504216168 0 0 0 0 0 3.931253 0 3.703704 12083 12083 12083 0 0 0 0 0 4.9312530 Control Cell 17544 Blank 0 Dilution 3 IC50 FALSE Inhibition effect toHBsAg 300 9226 8196 9658 0.152489 0.247106 0.521054 0.1708 0.1892950.8292 0.185857736 100 10946 10340 10828 0 0.050156 0.364272 0.0184950.0184947 1.810705 0.010110817 33.33333 12250 12980 13934 0 0 0 0 02.810705 0 11.11111 12634 12342 12000 0 0 0 0 0 3.810705 0 3.70370410886 10886 10886 0 0 0 0 0 4.810705 0 Control Cell 10886 Blank 0Dilution 3 IC50 FALSE

TABLE 3 Results of inhibition rate of Infergen ® to HBsAg and HBeAgInhibition rate Average Accumulated Concentration First Second ThirdFirst Second Third inhibition inhibition (×10⁴IU/ml) well well well wellwell well rate Accumulation 1-Accumulation rate First batch:(Infergen ®) Inhibition effect to HBeAg 900 14172 12156 17306 0.0916550.220869 0 0 0.104175 0.306157 0.895825 0.254710274 300 13390 1228816252 0.1417767 0.212409 0 0 0.118062 0.2019827 1.777764 0.102024519 10014364 18834 14194 0.079349 0 0.090245 0.056531 0.083921 2.7212320.029916678 33.33333 15722 16034 16340 0 0 0 0 0.0273897 3.7212320.007306592 11.11111 17504 17652 14320 0 0 0.082169 0.02739 0.027394.693843 0.005801377 Control Cell 15602 Blank 0 Dilution 3 IC50 FALSEInhibition effect to HBsAg 900 12080 11692 12234 0 0.01275 0 0.004250.025163 0.99575 0.024647111 300 12840 11484 12350 0 0.030313 0 0.0101040.0209125 1.985646 0.010422073 100 12894 14696 15086 0 0 0 0 0.0108082.985646 0.003606955 33.33333 15032 12928 13020 0 0 0 0 0.01080813.985646 0.002704416 11.11111 11794 11984 11508 0.004137 0 0.0282870.010808 0.010808 4.974837 0.002167838 Control Cell 11843 Blank 0Dilution 3 IC50 FALSE Second batch: (Infergen ®) Inhibition effect toHBeAg 900 6278 6376 6408 0.200051 0.187564 0.183486 0.190367 0.2746350.809633 0.253290505 300 7692 9092 6394 0.0198777 0 0.18527 0.0683830.0842678 1.74125 0.046161005 100 8960 7474 8190 0 0.047655 0 0 0.0158850.015885 2.725365 0.005794856 33.33333 8530 8144 9682 0 0 0 0 0 3.7253650 11.11111 7848 7848 7848 0 0 0 0 0 4.725365 0 Control Cell 7848 Blank 0Dilution 3 IC50 FALSE Inhibition effect to HBsAg 900 12364 12268 122740.036171 0.043655 0.043187 0.041004 0.140162 0.958996 0.12751773 30011590 12708 13716 0.0965076 0.009355 0 0.035287 0.0991581 1.9237090.0490186 100 12448 13468 13982 0.029623 0 0 0.009874 0.063871 2.9138340.02144964 33.33333 12616 11346 12444 0.016526 0.115529 0.0299350.053996 0.0539965 3.859838 0.013796309 11.11111 12828 12828 12828 0 0 00 0 4.859838 0 Control Cell 12828 Blank 0 Dilution 3 IC50 FALSE Thirdbatch: (Infergen ®) Inhibition effect to HBeAg 900 7240 6642 61580.064599 0.14186 0.204393 0.136951 0.217399 0.863049 0.201211735 30011072 8786 6902 0 0 0.108269 0.03609 0.0804479 1.82696 0.042176564 1007016 9726 7552 0.09354 0 0.024289 0.039276 0.044358 2.787683 0.01566301733.33333 7622 8866 8676 0.015245 0 0 0.005082 0.0050818 3.7826010.001341671 11.11111 7740 7740 7740 0 0 0 0 0 4.782601 0 Control Cell7740 Blank 0 Dilution 3 IC50 FALSE Inhibition effect to HBsAg 900 1104811856 11902 0.04775 0 0 0.015917 0.015917 0.984083 0.015916796 300 1345412896 11798 0 0 0 0 0 1.984083 0 100 12846 13160 12546 0 0 0 0 02.984083 0 33.33333 12680 12458 12360 0 0 0 0 0 3.984083 0 11.1111111602 11602 11602 0 0 0 0 0 4.984083 0 Control Cell 11602 Blank 0Dilution 3 IC50 FALSE HBeAg: Average IC50: 0 SD: 0 HBsAg: Average IC50:0 SD: 0

Example 5 Preparation of rSIFN-co

Preparation of lyophilized injection Lyophilized powder Stock Solutionof 34.5 μg/ml rSIFN-co PB (pH 7.0) 10 mmol/L Glycine 0.4 mol/L

Preparation technique: Weigh materials according to recipe. Dissolvewith sterile and pyrogen-free water. Filter through 0.22 μm membrane tode-bacterialize, preserve at 6-10° C. Fill in vials after affirming itis sterile and pyrogen-free, 0.3 ml/vial or 0.5 ml/vial, and lyophilizein freeze dryer.

Preparation of liquid injection Solution Stock Solution of 34.5 μg/mlrSIFN-co PB (pH 7.0) 25 mmol/L NaCl 0.1 mol/L

Preparation: Weigh materials according to recipe. Add to desired levelwith sterile and pyrogen-free water. Filter through 0.22 μm membrane tode-bacterialize, preserve at 6-10° C. Fill in airtight vial afteraffirming it is sterile and non-pyrogen at 0.3 ml/vial or 0.5 ml/vial.Storage at 2-10° C., and protect from light.

Example 6 Acute Toxicity of rSIFN-co

Treat mice with large dose (150 μg/kg, equal to 1000 times of the normaldose per kilo used in treatment of adult patients) of rSIFN-co at onetime by intramuscular injection. Then, observe and record their deathsand toxic reactions. Results show that: 24 hours after injection, noabnormal reaction had been recorded. The organs of the animals which hadbeen selected to be killed also had no signs of abnormal changes. Thoseremaining mice were all kept alive and were normal after two weeks. Theweights of mice in the experimental group and control group allincreased, and the ratio of increase had no obvious difference betweenthe two groups (P>0.05) according to their weights on the fourteenthday. No abnormal changes were seen from the main organs of those miceafter two weeks.

1. Experimental material

1.1 Animals

40 healthy adult mice, weighing 18-22 g, half male and half female,qualified by Sichuan experiment animal control center.

2.2 Medicines

rSIFN-co (Provided by Sichuan Huiyang Life-engineering Ltd.) sterilizedsolution, 0.15 mg/ml, Lot: 981201

rSIFN-co was administered i.m. in saline.

2. Method

Separate the 40 mice into two groups randomly, one for experimentalmedicine, another for control. Inject medicines or saline at the sameratio (0.1 ml/10 g) through muscle to each mouse according to whichgroup they belong. (150 μg/kg of rSIFN-co for experimental group; andsaline for control group). After injection, observe and record acutetoxicity shown in mice. Kill half of the mice (male and female eachhalf) to check whether there were any abnormal pathologic changes intheir main organs, such as heart, spleen, liver, lung, kidney, adrenalgland, stomach, duodenum, etc. after 24 hours. Those remains were keptand observed until the fourteenth day. Weigh all mice, kill them, andthen observe the appearance of the organs listed above to see if thereare any abnormalities. Take pathological tissue and examine it, usingthe examination to assess the difference in weight increases in the twogroups.

3. Results

Results show that there was no acute toxicity seen after all mice weretreated with i.m. rSIFN-co with 150 μg/kg at a time, equal to 1000 timesthe normal dose per kilo used in treatment of adult patients. In the 14days after injection, all mice lived well. They ate, drank, exercised,and excreted normally and showed normal hair conditions. None of themdied. The observation of the main organs of the randomly selected miceshows no abnormal changes 24 hours after injection. 14 days afterinjection, all remaining mice were killed. Autopsies also showed nochanges. The weights of mice in the two groups all increased, but noobvious difference was shown when accessed with statistic method(p>0.05). See Table 1:

TABLE 1 Influence to weights of mice after injection of rSIFN-co WeightsWeights Increased before after value of injection injection weightsGroup Dose Animal (g) (g) (g) Control 0 20 19.8 ± 1.7 30.8 ± 2.8 11.0 ±2.9 rSIFN-co 150 20 19.4 ± 1.7 32.1 ± 3.3 12.7 ± 4.3

3. Conclusion

Under conditions of this experiment, there were no toxic reactions inall mice after injection of rSIFN-co with 150 μg/kg. The conclusion canbe reached that the maximum tolerable dose of i.m. in mice is 150 μg/kg,which is equal to 1000 times the normal dose per kilo used in treatmentof adult patients.

Example 7 The Clinic Effects of Recombinant Super-Compound Interferon(rSIFN-co)

The recombinant super-compound interferon (rSIFN-co) is an invention forviral disease therapy, especially for hepatitis. Meanwhile, it caninhibit the activity of EB viruses, VSV, Herpes simplex viruses,cornaviruses, measles viruses et al. Using Wish cells/VSV system as theassay for anti-virus activity, the results showed that: the other rIFN,was 0.9×10⁸ IU/mg, Intron A was 2.0×10⁸ IU/mg and rSIFN-co was 9×10⁸IU/mg. The anti-viral activity of rSIFN-co is much higher than those ofthe former two.

Under the permission of the State Food and Drug Administration (SFDA),People's Republic of China, the clinical trials have taken place in WestChina Hospital, Sichuan University, the Second Hospital of ChongqingMedical University, the First Hospital of School of Medical, ZhejiangUniversity since the February 2003. The clinical treatment which focuseson the hepatitis B is conducted under the guidance of the mutilcenter,double-blind random test. IFN-α1b was used as control, and the primaryresults showed the following:

The Effect of rSIFN-co Compared with IFN-α1b in the Treatment of ChronicActive Hepatitis B

1. Standard of patients selection: The standard 1-4 are effective toboth treatment with rSIFN-co (9 μg) and IFN-α1b (5 MU, 50 μg), and thestandard 1-5 are for rSIFN-co (15 μg) treatment.

1). Age: 18-65

2). HBsAg test positive last over six months, HBeAg test positive, PCRassay, HBV-DNA copies 10⁵/ml

3). ALT≧two times of the normal value

4). Never received IFN treatment; or those received the Lamividinetreatment but failed or relapsed

5) Once received other IFNs (3MU or 5MU) treatment six months ago,following the standard of SFDA but failed or relapsed

2. Evaluation of the Effects:

In reference to the recommendations from the Tenth China NationalCommittee of Virus Hepatitis and Hepatopathy, the effects were dividedinto three degrees according to the ALT level, HBV-DNA and HBeAg tests.

-   Response: ALT normal level, HBV-DNA negative, HBeAg negative-   Partial response: ALT normal level, HBV-DNA or HBeAg negative-   Non response: ALT, HBV-DNA and HBeAg unchanged-   The response and partial response groups consider as effective    cases.

3. Results of Clinic Trial:

HBsAg HBeAg HBV-DNA Transfer Transfer Transfer Heptal to to to functionEffective negative negative negative Recover Period group Medicine casesRate rate rate rate rate 8-12 A rSIFN- 32 46.88 9.38 28.12 37.50 84.38week co (9 μg) (15) (3) (9)  (12) (27) B IFN-α1b 32 21.88 0.00 9.3815.62 56.25 (5 MU, (7)  (0) (3)  (5)  (18) 50 μg) 16-24 A rSIFN- 6454.69 7.81 25.00 34.38 90.62 week co (9 μg) (35) (5) (16) (22) (58) BIFN-α1b 64 25.00 0.00 9.38 18.75 78.13 (5MU, (16) (0) (6)  (12) (50) 50μg) Group A: treatment with rSIFN-co (9 μg) Group B: treatment withIFN-α1b (5MU, 50 μg)

In Group C, the cases were chronic active hepatitis B treatment withother IFNs (3MU or 5MU) before but failed or relapsed and treated withrSIFN-co (15 rag), subcutaneous injection, every one day, last 24 weeks.The total cases are 13. After 12 weeks treatment, 7 of 13 (53.85%) wereeffective. 3 of 13 (23.08%) HBeAg transferred to negative; 7 of 13(53.85%) HBV-DNA transferred to negative; 11 of 13 (84.62%) hepalfunctions recovered to normal.

4. The Side Effects of rSIFN-co Compared with IFN-α1b in the Treatment

The side effects of IFN include fever, nausea, myalgia, anorexia, hairlose, leucopenia and thrombocytopenia, etc. The maximum dose of IFN-α1bis 5MIU per time; the routine dose is 3 MIU. When taken the routinedose, 90% patients have I-II degree (WHO standard) side effects. Theyare fever lower than 38° C., nausea, myalgia, anorexia, etc. When takenat maximum dose, the rate of side effects do not rise obviously, but aremore serious. The maximum dose of rSIFN-co is 24 μg, subcutaneousinjection, every one day for 3 months. The routine dose is 9 μg. Whenroutine doses were used, less than 50% patients have I-II degree (WHOstandard) side effects, including fever below 38° C., nausea, myalgia,anorexia, leucopenia and thrombocytopenia slightly. With maximum dosage,about 50% patients suffered from leucopenia and thrombocytopenia afterusing rSIFN-co one month, but those side effects would disappear afterstopping treatment for one week. It is safe for continue use.

The Observations of rSIFN-co Treat Hepatitis C

1. Standard of Patient's Selection

1) age: 18-65

2) HCV antibody positive

3) ALT≧1.5 times of the normal value, last more than 6 months

2. Evaluation of the Effects:

Referring to the standard of Infergen® for treatment of hepatitis C andaccording to the ALT level and HCV-RNA test, divided the effects intothree degree:

Response: ALT normal level, HCV-RNA negative

Partial response: ALT normal level, HCV-RNA unchanged

Non response: ALT and HCV-RNA unchanged

3. Effects in Clinic

The clinical trial was done at the same time with hepatitis B treatment.46 cases received the treatment, 9 μg each time, subcutaneous injection,every day for 24 weeks. After treatment, 26 of 46 (56.52%) have obviouseffects, 12 of 46 (26.08%) HCV-RNA transferred to negative, 26 of 46(56.52%) hepal functions recovered to normal.

1. A recombinant super-compound interferon or a functional equivalentthereof with changed spatial configuration and improved efficacy.
 2. Theinterferon of claim 1, wherein the interferon is either α, β, or ω. 3.The interferon of claim 1, wherein the interferon has higher efficacythan the interferon described in U.S. Pat. No. 4,695,623 or 4,897,471.4. A super-compound interferon of claim 1 with unique secondary ortertiary structure.
 5. The super-compound interferon of claim 1, whereinthe 3-dimensional change is the result of changes of its productionprocess.
 6. A super-compound interferon of claim 1, produced by a highefficiency expression system which uses a special promoter.
 7. Thesuper-compound interferon of claim 6, wherein the promoter is P_(BAD).8. The super-compound interferon of claim 5, wherein its gene isartificially synthesized cDNA with adjustment of its sequence from thewild-type according to codon preference of E. coli.
 9. Thesuper-compound interferon of claim 1, which possesses anti-viral oranti-tumor activity.
 10. (canceled)
 11. The super-compound interferon ofclaim 10, which directly inhibits the DNA duplication and secretion ofHBsAg and HBeAg of Hepatitis B Virus.
 12. An artificial gene codes forthe super-compound interferon or its equivalent of claim
 1. 13. A vectorcomprising the gene of claim
 12. 14. An expression system comprising thevector of claim
 13. 15. A host cell comprising the vector of claim 13.16. A process for production of recombinant super-compound interferoncomprising introducing an artificial gene with selected codon preferenceinto an appropriate host, culturing said introduced host in anappropriate condition for the expression of said compound interferon andharvesting the expressed compound interferon. 17-22. (canceled)
 23. Acomposition comprising the recombinant super-compound interferon ofclaim 1 and a suitable carrier.
 24. A pharmaceutical compositioncomprising the recombinant super-compound interferon of claim 1 and apharmaceutically acceptable carrier.
 25. A method for preventing ortreating viral diseases or tumor in a subject comprising administeringto the subject an effective amount of the super-compound interferon ofclaim
 1. 26. (canceled)
 27. The method of claim 25 whereinsuper-compound interferon was administered via oral, vein injection,muscle injection, peritoneal injection, subcutaneous injection, nasal,mucosal administration, by inhalation via an inspirator.
 28. The methodof claim 25 wherein super-compound interferon was administered followingthe protocol of injection 9 μg or 15 μg per day, 3 times a week, total24 weeks.